Multiplexed methylation detection methods

Abstract

The present invention is directed to sensitive and accurate multiplexed assays for target analyte detection and detection of methylation in nucleic acid samples.

What is claimed is: 
     
       1. A method of detecting methylation of a cytosine in a target nucleic acid sequence in a sample of nucleic acids, comprising:
 a) contacting the sample of nucleic acids with bisulfite and forming treated nucleic acids, wherein non-methylated cytosines of the target nucleic acid sequences of the treated nucleic acids are converted to uracil, wherein methylated cytosines of the target nucleic acid sequences of the treated nucleic acids are not converted to uracil, and wherein the target nucleic acid sequences of the treated nucleic acids comprise locus sequences and either cytosines or uracils at the potentially methylated positions of the target nucleic acid sequences of the treated nucleic acids; 
 b) contacting said treated nucleic acids in a single reaction with a population of single-stranded first probes and a population of single-stranded second probes, wherein the first probes are complementary to locus sequences and uracils at the potentially methylated positions of the target nucleic acid sequences of the treated nucleic acids and each probe in said population of first probes comprises an identical first universal priming site, and wherein the second probes are complementary to locus sequences and cytosines at the potentially methylated positions of the target nucleic acid sequences of the treated nucleic acids and each probe in said population of second probes comprises an identical second universal priming site, thereby forming first double-stranded hybridization complexes comprising the first probes and second double-stranded hybridization complexes comprising the second probes, respectively; 
 c) contacting said first and second hybridization complexes with a polymerase or a ligase that modifies the first and second probes of the first and second hybridization complexes and forming single-stranded, first and second modified probes, respectively; 
 d) contacting said first and second modified probes with a composition comprising a polymerase, dNTPs, and at least first and second universal amplification primers that are complementary to said first and second universal priming sites, respectively, wherein said first and second modified probes are amplified and forming first and second amplicons, respectively; and 
 e) detecting said first and second amplicons, such that detection of said first amplicons indicates the presence of a non-methylated cytosine in the target nucleic acid sequence in the sample of nucleic acids, and detection of said second amplicon indicates the presence of a methylated cytosine in the target nucleic acid sequence in the sample of nucleic acids. 
 
     
     
       2. The method according to  claim 1 , wherein said first and second modified probes are amplified in step d) by a method selected from the group consisting of polymerase chain reaction (PCR)-oligonucleotide ligation assay (OLA), polymerase chain reaction (PCR) and rolling circle amplification (RCA). 
     
     
       3. The method according to  claim 2 , wherein said first and second modified probes are amplified by polymerase chain reaction (PCR). 
     
     
       4. The method according to  claim 1 , wherein said first and second amplicons are detected by hybridizing said first and second amplicons to an array. 
     
     
       5. The method according to  claim 4 , wherein said array is selected from the group consisting of an ordered array, a liquid array and a random array. 
     
     
       6. The method according to  claim 1 , wherein each of said first or second probes further comprises an adapter sequence, whereby the adapter sequence remains present in the first or second modified probes in step c). 
     
     
       7. The method according to  claim 6 , wherein said first or second modified probes in step d) are detected by detecting said adapter sequence of each of said first or second modified probes. 
     
     
       8. The method according to  claim 7 , wherein said detecting said adapter sequence comprises hybridizing said adapter sequence with a capture probe that is complementary to said adapter sequence. 
     
     
       9. The method according to  claim 1 , wherein step b) further comprises further contacting said treated nucleic acids with a downstream probe comprising a sequence substantially complementary to a sequence of the treated target nucleic acid sequence. 
     
     
       10. The method according to  claim 9 , wherein each of said first and second probes in the first and second hybridization complexes is adjacent to the 5′ end of the downstream probe and is contacted in step c) with only a ligase, whereby each of said first and second probes is ligated to the downstream probe, resulting in the first and second modified probes. 
     
     
       11. The method according to  claim 10 , wherein the downstream probe further comprises an adapter sequence, whereby the adapter sequence remains present in the first and second modified probes in step c). 
     
     
       12. The method according to  claim 9 , wherein each of said first and second probes in the first and second hybridization complexes is adjacent to the 5′ end of the downstream probe and said first and second hybridization complexes are contacted in step c) with a polymerase and a ligase in the presence of nucleotides, whereby at least one of the nucleotides is added to the first and second probes, whereby each of said first and second probes is ligated to the downstream probe, resulting in the first and second modified probes. 
     
     
       13. The method of  claim 9 , wherein each of said first and second probes in the first and second hybridization complexes is adjacent to the 5′ end of the downstream probe and step c) comprises ligation of each of the first probes of the first hybridization complex or each of the second probes of the second hybridization complex to the downstream probe. 
     
     
       14. The method of  claim 9 , wherein the downstream probe further comprises a downstream priming site. 
     
     
       15. The method according to  claim 1 , wherein said first and second hybridization complexes are contacted in step c) with only a polymerase in the presence of nucleotides, whereby at least one of the nucleotides is added to the first and second probes, resulting in the first and second modified probes. 
     
     
       16. The method according to  claim 1 , wherein the population of the first probes or the second probes comprises more than 10 different probes. 
     
     
       17. The method according to  claim 1 , wherein the population of the first probes or the second probes comprises more than 1000 different probes. 
     
     
       18. The method according to  claim 1 , wherein the target nucleic acid sequence comprises a CpG site. 
     
     
       19. The method according to  claim 18 , wherein the CpG site is within a CpG island locus. 
     
     
       20. The method according to  claim 1 , wherein the composition in step d) further comprises a downstream amplification primer. 
     
     
       21. The method according to  claim 1 , wherein each of said first or second probes further comprises an adapter sequence, whereby the adapter sequence remains present in the first or second modified probes in step c), and when said first and second modified probes are amplified and forming the first and second amplicons in step d), each of said first or second amplicons thereby comprises said adapter sequence. 
     
     
       22. The method according to  claim 21 , wherein detecting the first or second amplicons comprises detecting said adapter sequence by hybridization with a capture probe that is complementary to said adapter sequence. 
     
     
       23. The method according to  claim 21 , wherein detecting the first or second amplicons comprises detecting said adapter sequence of each of said first or second amplicons. 
     
     
       24. The method according to  claim 23 , wherein said detecting said adapter sequence comprises hybridizing said adapter sequence with a capture probe that is complementary to said adapter sequence. 
     
     
       25. The method according to  claim 1 , wherein the first amplicons are labeled amplicons when the first universal primers are labeled. 
     
     
       26. The method according to  claim 1 , wherein the second amplicons are labeled amplicons when the second universal primers are labeled. 
     
     
       27. The method according to  claim 1 , wherein the first amplicons are labeled amplicons when one or more of the dNTPs are labeled. 
     
     
       28. The method according to  claim 1 , wherein the second amplicons are labeled amplicons when one or more of the dNTPs are labeled.